The present invention concerns a nucleic acid which is capable of regulating the transcription of the ABC1 gene, which is a causal gene for pathologies linked to a dysfunctioning of cholesterol metabolism, inducing diseases such as atherosclerosis.
The invention also relates to nucleotide constructs comprising a polynucleotide which encodes a polypeptide or a nucleic acid of interest, placed under the control of a regulatory nucleic acid for the ABC1 gene.
The invention also relates to recombinant vectors, transformed host cells and non-human transgenic mammals comprising a nucleic acid which regulates the transcription of the ABC1 gene or an above-mentioned nucleotide construct, as well as to methods for screening molecules or substances which are capable of modifying the activity of the regulatory nucleic acid for the ABC1 gene.
The invention also relates to methods for detecting an impairment in the transcription of the ABC1 gene in an at-risk individual.
A subject of the invention is also substances or molecules which modify the activity of the nucleic acid which regulates the transcription of the ABC1 gene, as well as pharmaceutical compositions containing such substances or such molecules.
High density lipoproteins (HDLs) are one of the four major classes of lipoprotein which circulate in the blood plasma.
These lipoproteins are involved in various metabolic pathways, such as lipid transport, bile acid formation, steroidogenesis or cell proliferation, and also interfere with plasmatic proteinase systems.
HDLs are perfect free cholesterol acceptors, and in combination with cholesterol ester transfer proteins (CETP), lipoprotein lipase (LPL), hepatic lipase (HL) and lecithin-cholesterol acyltransferase (LCAT), play a major role in the reverse transport of cholesterol, i.e. the transport of excess cholesterol in the peripheral cells to the liver, for its removal from the body in the form of bile acid.
It has been demonstrated that the HDLs generally play a central role in the transport of cholesterol from the peripheral tissues to the liver.
Various diseases linked to an HDL deficiency have been described, including Tangier disease, HDL deficiency and LCAT deficiency.
The deficiency involved in Tangier disease is linked to a cellular defect in the translocation of cellular cholesterol, which leads to a degradation of the HDLs.
In Tangier disease, this cellular defect leads to a disruption of lipoprotein metabolism. The HDL particles in Tangier disease, which do not incorporate cholesterol from the peripheral cells, and which are not able to be correctly metabolized, are rapidly eliminated from the body. The plasma HDL concentration in these patients is thus extremely reduced, and the HDLs no longer contribute to the return of cholesterol to the liver. This cholesterol accumulates in these peripheral cells and causes characteristic clinical manifestations such as the formation of orange-colored tonsils. Furthermore, other lipoprotein disruptions such as an overproduction of triglycerides and an increased synthesis and intracellular catabolism of phospholipids are generally observed.
Tangier disease, the symptoms of which have been described above, is classified among the familial conditions linked to metabolism of the HDLs which are commonly detected in patients affected by coronary diseases.
Numerous studies have shown that a reduced level of HDL cholesterol is a risk factor which is useful for detecting a coronary condition.
In this context, syndromes linked to HDL deficiencies have been of increasing interest for the past decade, since they make it possible to increase the understanding of the role of HDLs in atherogenesis.
Several mutations in the apo A-1 gene have been characterized. These mutations are rare and can lead to an absence of production of apo A-1.
Mutation in the genes encoding lipoprotein lipase (LPL) or its activator apoC-II are associated with severe hypertriglyceridemias and substantially reduced levels of HDL-c.
Mutations in the gene encoding the enzyme lecithin-cholesterol acyltransferase (LCAT) are also associated with severe HDL deficiency.
Furthermore, dysfunctions in the reverse transport of cholesterol might be induced by physiological deficiencies affecting at least one of the steps for transporting stored cholesterol from the intracellular vesicles toward the membrane surface, where it is accepted by the HDLs.
Recently, a study was carried out on the segregation of various allelic forms of 343 microsatellite markers distributed over the entire genome and distant from each other by 10.3 cM on average.
The linkage study was carried out on a family which had been well characterized over eleven generations, in which many members are affected by Tangier disease, the family comprising five consanguineous lines.
This study made it possible to identify a region located in the 9q31 locus of human chromosome 9 which is statistically linked to the condition (Rust S. et al., Nature Genetics Vol. 20, September 1998, pages 96-98).
However, the study by Rust et al. only characterizes a wide region of the genome in which impairments are likely to be associated with Tangier disease. The study simply stated that the relevant 9q31-34 region contains ESTs, but no known gene.
It has been shown that a region spanning 1 cM, situated in the 9q31 locus in humans, is generally associated with familial HDL deficiencies (Rust et al., 1999).
Furthermore, it has been shown that a gene encoding a protein of the family of ABC transporters, which is located precisely in the 1 cM region of the 9q31 locus, is involved in pathologies linked to a deficiency in the reverse transport of cholesterol.
For example, it has been shown that the gene encoding the ABC-1 transporter is mutated in patients with affected reverse transport of cholesterol, such as in patients suffering from Tangier disease.
The ABC (“ATP-binding cassette”) transporter proteins constitute a family of proteins which are extremely conserved in evolution, from bacteria to humans.
The ABC transporter proteins are involved in the membrane transport of various substrates, for example, ions, amino acids, peptides, sugars, vitamins or steroid hormones.
The characterization of the complete amino acid sequence of some ABC transporters has made it possible to determine that these proteins have a common general structure, for example, two nucleotide-binding folds (Nucleotide Binding Fold or NBF) with moieties of Walker A type and Walker B type, as well as two transmembrane domains, each of the transmembrane domains consisting of six helices. The specificity of the ABC transporters for the various transported molecules appears to be determined by the structure of the transmembrane domains, whereas the energy required for the transport activity is provided by degrading ATP at the NBF fold.
Several of the ABC transporter proteins which have been identified in humans have been associated with various diseases.
For example, cystic fibrosis is caused by mutations in the CFTLR (cystic fibrosis transmembrane conductance regulator) gene.
Moreover, some multi-drug resistance phenotypes in tumor cells have been associated with mutations in the gene encoding the MDR (multi-drug resistance) protein which also has an ABC transporter structure.
Other ABC transporters have been associated with neuronal and tumor conditions (U.S. Pat. No. 5,858,719), or potentially implicated in diseases caused by an impairment of the homeostasis of metals, for example, the ABC-3 protein.
Similarly, another ABC transporter, referred to as PFIC2, seems to be involved in a form of progressive familial intrahepatic cholestasis, this protein being potentially responsible, in humans, for the export of bile salts.
In 1994, a cDNA encoding a novel mouse ABC transporter was identified and referred to as ABC1 (Luciani et al., 1994). This protein is characteristic of the ABC transporters in that it has a symmetrical structure comprising two transmembrane domains linked to a highly hydrophobic segment and to two NBF moieties.
In humans, a partial cDNA comprising the entire open reading frame of the human ABC1 transporter has been identified (Lanigmann et al., 1999).
It has also been shown that the gene encoding the human ABC1 protein is expressed in various tissues, and more particularly at high levels in the placenta, the liver, the lungs, the adrenal glands and the fetal tissues.
These authors have also shown that the expression of the gene encoding the human ABC1 protein is induced during the differentiation of monocytes into macrophages in vitro. Furthermore, the expression of the gene encoding the ABC1 protein is increased when human macrophages are incubated in the presence of acetylated low-density lipoproteins (AcLDLs).
The work of Rust S. et al., 1999, Brooks-Wilson A. et al., 1999, Bodzioch M. et al., 1999, Remaley A. et al., 1999 and of Marcil M. et al., 1999 has shown that patients suffering from Tangier disease and from HDL deficiencies (FHD; familial HDL deficiency) have a mutated ABC1 gene. Several mutations, which are distributed in various regions of the ABC1 gene, have been identified in the genome of various patients, for example, of patients with a severe form of the disease associated with coronary disorders. Moreover, diverse polymorphisms have been found, both in the exons and in the introns of the ABC1 gene, in patients suffering from milder forms of the disease, indicating that these patients carry specific alleles of the gene, which are distinct from the “wild-type” allele(s).
Although the expression of the human ABC1 gene seems to be regulated according to the type of cell or to the metabolic situation of a given cell type, the sequence(s) which make(s) it possible to regulate this gene were not known.
Thus, there exists a need in the state of the art to identify these regulatory sequences, for the two principal reasons below:
a) These sequences are likely to be mutated in patients suffering from a pathology linked to a deficiency in cholesterol transport, for example, in patients suffering from Tangier disease, or likely to develop such pathologies.
The characterization of the regulatory sequences of the human ABC1 gene would make it possible, firstly, to detect mutations in patients, and, for example, also to diagnose the individuals who belong to at-risk familial groups. In addition, the isolation of these regulatory sequences would make it possible to complement the mutated sequence with a functional sequence capable of overcoming the metabolic dysfunctions induced by the mutation(s) diagnosed, through the construction of targeted therapeutic means, such as means intended for gene therapy.
b) The characterization of the regulatory sequences of the ABC1 gene would place at the disposal of persons skilled in the art means capable of allowing the construction, by genetic engineering, and then the expression, of given genes in the cell types in which the ABC1 gene is expressed.
c) Moreover, some portions of the regulatory sequences of the ABC1 gene might constitute high expression-level constitutive promoter sequences, which are liable to enable the construction of novel means for expressing given sequences in cells, completing an already existing set of means.
To date, despite the efforts undertaken, the regulatory sequences of the ABC1 gene have remained totally unknown.
The inventors have henceforth isolated and then sequenced a genomic DNA comprising the first two exons of the ABC1 gene (respectively exon 1A and exon 1B), as well as a non-transcribed region of approximately 2.9 kb, which is located on the 5′ side of exon 1A, and which comprises regulation signals for the ABC1 gene.